The present invention relates to graft copolymers of crosslinked polymers and linear polyoxyethylene, processes for their production, and their use.
Graft copolymers of crosslinked, insoluble polymers and polyoxyethylene are of importance as substrates for peptide synthesis and for the immobilization of low-molecular and high-molecular active agents for affinity chromatography, diagnostic agents, and biotechnological methods. Heretofore, such graft copolymers have been prepared from crosslinked, chloromethylated polystyrene and shorter polyethylene glycols in accordance with the Williamson ether synthesis: ##STR1## (cf. Makromol. Chem. Rapid Commun. 3 : 217 [1982]; 2, 621 [1981]). One disadvantage of this process resides in that the polystyrene is frequently inadequately charged with polyoxyethylene. The yields drop very drastically, primarily with an increasing molecular weight of polyoxyethylene, and only relatively short oligoethylene glycol chains with molecular weights of up to 1320 could be bound to the polystyrene. Another drawback in the ether synthesis is the formation of cyclic ethers by the reaction of both terminal hydroxy groups of polyoxyethylene with the chloromethylated polystyrene whereby the terminal hydroxy groups, required for the carrier function, are once again decreased.
The graft copolymers produced in this way exhibit, in their usage, inadequate reactivity, a charging with polyoxyethylene that is too low, and an insufficient stability of the bond during immobilization. Therefore, linear, homogeneously soluble polymers, such as polyoxyethylene, have frequently been employed for peptide synthesis. These soluble polymers, though, can be separated only with extreme difficulty.
It is thus an object of the present invention to provide graft copolymers exhibiting higher reactivity, higher charging, and higher stability of the bond during immobilization than conventional polymers, as well as a process for producing these graft copolymers, which avoids the disadvantages of the above-described prior art process.
This object is obtained by the graft copolymers of the present invention, containing, on a crosslinked polymer, several polyoxyethylene residues or chains with an average molecular weight of 500-50,000, and having 0.02-15 meq free hydroxy groups per gram of copolymer. Preferably, the amount of hydroxy groups is 0.05-15 meq/g, most preferably 0.05-10 meq/g.
With the use of crosslinked polystyrenes, this range is preferably 0.02-2 meq/g, especially preferably 0.05-0.7 meq/g. When using polyvinyl alcohols as the crosslinked polymers, this range is 1-15 meq/g, preferably 1-10 meq/g.
The average molecular weight of t h e polyoxyethylene chains is preferably from 800-10,000, especially from 900 to 6,000 with the optimum range being from 2,000 to 3,000.
The crosslinked polymer is preferably a polyvinyl alcohol, polyhydroxystyrene, a polymer produced from chloromethylated polystyrene and ethylene glycol or oligoethylene glycol, or a polyacrylate or polymethacrylate functionalized by hydroxy groups. The extent of crosslinking of these polymers herein is generally 0.05-10%, preferably 0.1-8%, especially preferably 0.2-5%. The most suitable extent of crosslinking is 1-2%, especially when using polystyrenes crosslinked with divinylbenzene.
Binding of the polyoxyethylene chains to the crosslinked polymers takes place preferably by way of hydroxy or amino groups of the crosslinked polymer. These can be present per se in the polymer, such as, for example, in the polyvinyl alcohol and polyhydroxystyrene, or they can be introduced subsequently by functionalizing. The amount of hydroxy groups (extent of functionalization) is generally in a range from 0.02 to 25 meq/g of crosslinked polymer, preferably 0.05-15 meq/g. Most suitably, a polystyrene is utilized having an extent of functionalization of 0.05-0.7 meq/g, or a polyvinyl alcohol is utilized with an extent of functionalization of 1-15 meq/g.
The process for preparing the graft copolymers of the present invention is characterized by reacting crosslinked polymers with ethylene oxide.
By suitably choosing the reaction temperature, the reaction period, the monomer concentration, and the solvent, the reaction can be controlled so that any desired average molecular weight can be obtained for the polyoxyethylene chain. Preferably, the reaction temperature is in the range from 20.degree. to 100.degree. C., especially preferably in a range from 60.degree. to 80.degree. C. The reaction time is preferably 30 minutes to 150 hours.
The reaction medium employed is one of the aprotic, organic solvents inert to the reaction; ethers are especially suitable, such as, for example, dioxane, tetrahydrofuran, or diglycol ethers, as well as toluene, benzene, xylene, dimethylformamide, or dimethyl sulfoxide.
The reaction is optionally conducted in the presence of alkaline or acidic catalysts. Suitable alkaline catalysts are, for example, alkali metals, such as lithium, sodium, or potassium; metallic hydrides, such as sodium hydride, calcium hydride; alkali metal amides, such as sodium amide; Grignard compounds or alcoholates. Preferably, potassium is employed. Suitable acidic catalysts are, for example, hydrogen chloride, sulfuric acid, or p-toluenesulfonic acid.
Advantageously, in a first stage, oligoethylene glycol chains of the formula H--(OCH.sub.2 CH.sub.2).sub.n --OH, wherein n stands for 2-20, are bound to the crosslinked polymer. This reaction is carried out under conditions customary for etherification or Williamson synthesis. An aqueous sodium hydroxide solution can also serve as the base for the Williamson synthesis.
In a second stage, the oligoethylene chain is then extended with ethylene oxide. This two-stage process is suitable, in particular, for the production of polystyrene-polyoxyethylene graft copolymers.
The graft copolymers of the present invention can be utilized as substrates for peptide synthesis and nucleotide synthesis, for affinity chromatography, for the covalent fixation or immobilization of peptides, active protein compounds on enzymes in biotechnological reactions, and as active agents in diagnostic media.
On account of the hydroxy groups present in the graft copolymers of the present invention, peptides can be built up stepwise by means of conventional methods of peptide synthesis (Peptides, vol. 2, Academic Press, 1979). Surprisingly, such immobilized polyoxyethylenes with an average molecular weight of 1,000-2,000 show, in peptide coupling reactions, a higher reaction velocity than non-immobilized polyoxyethylenes in solution. This high reactivity thus also permits immobilization of proteins, enzymes, and other active compounds.
The degree of polymerization and/or the average molecular weight of the grafted copolymers can be affected by the parameters of temperature, time, and monomer concentration. For example, it has been found in connection with PSPOE (polystyrene-polyoxyethylene) that high degrees of polymerization cannot be attained at low reaction temperatures (56.degree.-58.degree. C.), in spite of high amounts of monomer added and a long reaction period. An average molecular weight is obtained for polyoxyethylene (POE) grafted onto a modified polystyrene substrate of 2,000 (PSPOE-2000).
Reaction temperatures that are too high, or polymerization velocities that are too high, lead to destruction of the polystyrene substrate matrix. A reaction temperature of 70.degree.-73.degree. C. proved to be favorable. Different degrees of polymerization can be obtained by varying the amounts of monomer added and the reaction time. The curve for PSPOE-5600 in FIG. 1 illustrates the course of the reaction with relatively low amounts of monomer added, while the curve for PSPOE-6900 illustrates the course of the reaction at higher amounts of added monomer. Data for the graft copolymers PSPOE are listed in Table 1, with the course of the reaction being shown in FIG. 1.